Research Article

Open Access Journal of Research Article Biomedical Science ISSN: 2690-487X

New Rhizobial of the Genus Fredii Entering Symbiosis with Plants

Bakhtiyor Umarov* Department of Natural Sciences, Uzbek State University of World Languages Tashkent, Uzbekistan

ABSTRACT

Sinorhizobium fredii

For the first time in the soils of the Tashkent region were isolated fast growing types of nodule bacteria of the genus from the soybean plants. Were studied of symbiotic properties of isolated microorganisms in sterile conditions with soybean plants. In the KEYWORDS:genome of the isolated Sinorhizobium strains was fredii found from 2 to 3 mega plasmids related only to these types of bacteria.

; Megaplasmid; Nitrogen fixation INTRODUCTION bacteria can live in the soil as saprophytes. The objective of these Rhizobium the formed studies is the selection of nodule bacteria from soybean plants. It is known that bacteria of the genus of MATERIALS AND METHODS timeof nodule they onproduce the roots several of certain physiologically of leguminous active plantssubstances, and fixing gave Glycine max). Large athe positive molecular effect nitrogen on the leguminousin the symbiosis plants. with Nodule the plant bacteria at the play same an In the Experiments, the plants of soybean ( genus rhizobium - is an aerobic gram-negative rods 0,7-1,8 mm pink nodules were collected from the different layers of the root important role in enriching the soil with nitrogen. Bacteria of the Tashkent region. For the isolation of pure cultures of soybean root long living in the soil and the plant surface. system of each plant during the phase of mass flowering plants, When infected bean cause of recent education of nodule roots nodule bacteria used method Israelskee [1] with some modifications formations. Penetrated to the root of a hair, they cause the active as described in the methodological recommendations. Purification nodulebacteria bacteria was performed used medium with 4x as reseeding described typical in the colonies methodological obtained exhausting stroke dilution method. For the growing culture of so-calledroot cell Bacteroides, division that most leads intense to the binding appearance molecular of nodules. nitrogen. Its Nodulenodule bacteriabacteria grow metabolize and become as ammonium thickened, salts,branched nitrates, forms amino - the manual [1]. acids, and other compounds. Micro vegetation experiments carried out in glass tubes For their carbon source can be mono- and disaccharides, size 3x25cm filled 1/3 of sterile o vermiculiteo under greenhouse some polysaccharides, organic acids, alcohols. On dense media conditions in a special artificial climate room with an air nodule bacteria form round colorless, transparent, slimy colonies, 16-hour photoperiod. Seeds temperature in the daytime 27-30 C,2 20-30 C night, 50% relative oC. Nodules formed active contain humidity, light intensity of 200W/m pigment leghemoglobin and therefore painted in pink. Different of soybean plants for micro vegetation experiments was sterilized nodulegrowing bacteria well at 25cause the development of nodules in certain prior to seeding with sulfuric acid and up to 5 minutes, and then leguminous plants: Rhizobium meliloti - alfalfa, clover; Rhizobium washed with sterile tap water to a pH of 7.0. Sterile seedso were leguminosarum - peas and broad beans; Rhizobium trifolii C placed in Petri dishes containing 1% agar, to maintain humidity. only for clover, Bradyrhizobium japonicum and Sinorhizobium Next was charged in a Thermostat at a temperature of 28 for 2-3 fredii for . After the destruction of nodule, the is specificnodule days. The shoots of the plants were placed in a glass tube 3x25cm in size, filled to 1/3 with sterile vermiculite and grown for 35-40 days Quick Response Code: Address for correspondence: Bakhtiyor Umarov, Department of Natural Sciences, Uzbek State University of World Languages Tashkent, Uzbekistan Received: April 07, 2021 Published: April 30, 2021

How to cite this article: Bakhtiyor U. New Rhizobial Bacteria of the Genus Sinorhizobium Fredii Entering Symbiosis with Soybean Plants. 2021- 3(6) OAJBS.ID.000273. DOI: 10.38125/ OAJBS.000273

C 2021 Open Access Journal of Biomedical Science 942 Open Acc J Bio Sci. April- 3(6): 942-944 Bakhtiyor Umarov Research Article

Sinorhizobium fredii isolate nodule bacteria of leguminous plants, the selected soybean in 6-fold replicates. Nitrogen-fixing ability of isolates field, where before grew legumes were sown for several years. To determined by acetylene [2] with a flame ionization detector gas chromatograph ЛХМ-80 (USSR) and expressed in nM. Morpho- plants were in the flowering - fruiting phase: well-developed plants physiological properties of selected bacterial isolates were inand the their materials roots and were methods. selected, dense, large, pink nodules were studied by standard methods [3]. Genetic analysis on the plasmid selected. Isolation of nodule bacteria was carried out as described preparations were performed according to the method Eckhardt [4]. To determine the ability of bacteria to absorb different types of carbohydrates as sole carbon source was used sucrose, glucose The isolates were plated on Petri dishes containing a special (concentration of carbohydrates in the bean liquid medium was singlenutrient colonies medium appeared and kept seededin a thermostat bacteria. at Within a temperature 7-10 days of 27-the 1%). Microscopic analysis of cell nodule bacteria was conducted on 28 °C. After 3 days of incubation in growth medium plates separate aRESULTS light microscope AND DISCUSSION“Olympus” (Japan). growth of the bacteria was observed. During this time, the soybean nodule bacteria acquired colorless color touch and a smooth consistency (Figure 1). The legume plants were growing in the soils of the experimental

Figure 1: The rhizobia cells.

According to the morphological type of the isolated bacteria circular DNA molecule. By electrophoresis of cell lysates using Rhizobium chromosomeclassified in theand group several of mega bacteria plasmids of the genuscomposed a very (rod-long the method of Eckhardt [4], we have found 3 mega plasmid with shaped with rounded ends). Genetic structure consisted of one dimensions of 1500, 1600, 1700kb with respect to only these types of bacteriacarbon nutrition (Figure 2). and environment MPA. After separation of the isolated strains of rhizobia soybeans on a certain kind, determined the time of appearance and size of cell colonies. Are plated into individual cups of the most typical strains studied colonies S. fredii S. fredii B. japonicum should form colonies on the. Fast-growing 7th strains of formed colonies on day 2-4, speciesand slow-growing S. fredii strains of day after sowing. Pure cultures of soybean rhizobia gave good growth on MRS media with mannitol and glucose. To characterize the growth of strains of rhizobia on various sources of carbon nutrition was determined by the color change of the medium from inoculation in comparison with the control tube. Symbiotic nitrogen-fixing properties and the ability of isolates tested in micro vegetation experiments. Micro vegetation Figure 2: Plasmid profiles ofS.fredii (1,2,3, S.ferdi -T1, experiments were carried out under greenhouse conditions in the S.ferdi -T2, S.ferdi -T3, 4. S.meliloti 41). laboratoryS. fredii in 2019-2020. 6-107 1ml of culture strains to each of tube. nodule Assimilation bacteria wereof molecular collected nitrogen in the rhizobialogarithmic acetylene growth phasemethod 10 has beencells/ml tested, and usedas described to inoculate in isolates S. ferdi -T1, S. ferdi S. ferdi of strainFigure S. 2 meliloti shows the results of detection of the plasmids these -T2, -T3 compared to the type S. fredii. At determination materials and methods. As a result of this work has been allocated 41 from the collection DSMC (Germany). To 3 strains of soybean nodule bacteria determine the species, isolates were plated on medium (MRS) with aspectual accessories nodule bacteria were founded on got result various components of carbon nutrition and plain agar (MPA). had to additional proof to earlier published publication about species B. japonicum and on the grounds of their specificity to plant master. Our results From the reference it is known that soybean root nodule bacteria grow well on MRS medium with mannitol and quickly - and slow-growing bacteria. Wilson [5] has shown that glucose, do not grow on media containing disaccharides (sucrose, bacteria species S. fredii bacteria’s rhizobia are divided on 2-greater groups fast-growing lactose, maltose) and meat peptone agar (MPA). Soybean nodule and slow-growing, sharply differing on sort of the features. The grow well on MRS media with all sources C 2021 Open Access Journal of Biomedical Science 943 Open Acc J Bio Sci.April- 3(6): 942-944 Research Article Research Article Bakhtiyor Umarov

REFERENCES similar results are received and beside other authors shown that 1. rhizobia sort S. fredii pertain to group fast growing bacteria and Novikova AT, Simarov BV, Arriam L (1979) The methodical Bradyrhizobiumenter in symbiosis japonicum with plants from plants of soybean of soybean [6]. There that, basically, is much recommendations on reception new strains nodule bacteria and scientific works. In literature, denoted isolation of nodule bacteria estimation their efficiency. p. 33. 2. Hardy RWF, Burns RC, Holsten RD (1973) Applications of the acetylene- plants to soybean enter in symbiosis with these bacteria [7]. ethylene assay for measurement of nitrogen fixation. Soil Biol Biochem of nodule bacteria from sort S. fredii meet in soils of the Dalney 5(1): 47-81. Keyser [8] with colleague have shown that fast-growing types 3. Egorova NS (1989) Industrial Microbiology. p. 688.

Vostok, (Russia) entering in symbiosis withRhizobium plants ofsp soybean. 4. Eckhardt T (1978) A rapid method for the identification of plasmid Khalid showed that peanut rhizobia on stressful soils are fast deoxyribonucleic acid in bacteria. Plasmid 1(4): 584-588. growingDISCUSSION plants and phylogenetically related [9]. 5. Wilson JK (1944) Over five hundred reasons for abandoning the cross- 6. From above stated result and from result conducted similar inoculation groups of legumes Soil Sci 58(1): 61-69. Michael HS, Gerald HE (1982) Rhizobium fredii sp nov a fast-growing B. species that effectively nodulates soybeans. International Journal of 7. japonicumwork and besideand S. fredii authors,. is shown that plants to soybean on its Systematic Bacteriology 34(1): 484-489. host specificity can enter in symbiosis of nitrogen-fixing bacteria Jordan DC (1982) Transfer of Brady rhizobium gen nov a genus of slow- growing, root nodule bacteria from leguminous plants. Int J Syst Evol 8. Bacteriol 32(1): 136-139. of theThereby, sort of from S. fredii presented from the given plant is seen soybean, that usOn for the the grounds first time of in ground Uzbekistan is chosen fast-growing type of nodule bacteria Keyser HH, Bohlod B, Hu TS, Weber DF (1982) Fast growing rhizobia isolated from the nodules of soybean. Science 215(4540): 1631-1632. bacteria to soybean can meet on territory Uzbekistan and in foregoing, possible draw a conclusion: fast-growing types of nodule 9. Rabia K, Xiao XZ, Rifat H, Mukhtar A (2020) Molecular characteristics of Rhizobia isolated from arachis hypogaea grown under stress environment. Sustainability 12(15): 6259. Central-Asia regions where cultivate the soybean plants.

C 2021 Open Access Journal of Biomedical Science 944 Open Acc J Bio Sci. April- 3(6): 942-944